We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by a single infected infected fly (Phlebotomus duboscqi). Most of the infected mice were inoculated with a low dose of fewer than 260 Leishmania parasites. However, one in four received a higher dose of up to 100,000. To test the impact of the size of the inoculum on disease outcome, we compared high (5000) and low (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly derived metacyclic forms of L. major and pre-exposed the injection site to the bites of un-infected flies. As expected, pathology was significantly worse in the mice receiving the higher dose, but unexpectedly, the number of persisting parasites following healing was comparable in the low and high dose challenged mice. The reservoir potential of mice with cutaneous leishmaniasis initiated by infected sand fly bites was explored by exposing their infected ears to uninfected flies and disecting the midguts of fed flies after 2-3 days to determine the proportion that had obtained an infective bloodmeal. The frequency of positive flies ranged from 5-40% throughout the course of the cutaneous infection, and healed lesions were as efficient a source of L. major as active lesions. More surprisingly, there was no correlation between the parasite burden in the infected ears and the frequency with which flies picked up parasites from these ears. The findings suggest that flies might feed selectively on localized tissue sites harboring infected cells, and/or that tissue amastigotes are heterogenous with respect to their ability to survive and differentiate into promastigotes in the vector midgut. [unreadable] The ability of phagocytic cells to rapidly localize to sites of tissue injury and subsequently capture and destroy pathogens is a hallmark of the innate immune response, highly conserved across vertebrate species. Few studies, however, have directly examined the inflammatory response initiated during transmission of an infectious pathogen by arthropod vectors and the resulting impact of this process on disease progression. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and were subsequently transferred to macrophages. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.major appears to have evolved to both evade and exploit the innate wound healing response to sand fly bite in order to establish infection and promote disease.[unreadable] Based on the strong linkage disequilibrium reported in several Leishmania species, it is widely accepted that these parasites are essentially clonal. This notion must be reconciled, however, with the accumulating examples of naturally occurring hybrid strains, which appear to share phenotypic and genotypic markers from two perceived species, providing circumstantial evidence for sexual recombination. Clearly, the clonality vs sexuality debate would be far better informed if Leishmania were actually shown to be capable of genetic exchange, evidence for which is lacking until now. Using parental lines bearing distinct drug resistant markers, we have explored the possibility of genetic exchange during the growth and development of L. major promastigotes in the midgut of a natural sandfly vector species, Phlebotomus duboscqi. The results provide the first conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle, generating both diploid and triploid progeny bearing hybrid genotypes that strongly suggest meiotic division and uniparental inheritance of maxicircle kDNA.